Lissoni P, Messina G, Pelizzoni F, Rovelli F, Brivio F, Monzon A, Crivelli N, Lissoni A, Tassoni S, Sassola A, Pensato S, Di Fede G. The Fascination of Immuno- logical Science. J Infectiology. 2020; 3(1): 18-28 Journal of Infectiology Journal of Infectiology

Review Article Open Access

The Fascination of Cytokine Immunological Science Paolo Lissoni1*, Giusy Messina1, Francesco Pelizzoni2, Franco Rovelli1, Fernando Brivio1, Alejandra Monzon1, Na- dal Crivelli1, Arianna Lissoni1, Simonetta Tassoni3, Andrea Sassola1, Sonja Pensato1, Giuseppe Di Fede1 1Institute of Biological Medicine, Milan, Italy 2Cardiological Surgery Division, Niguarda Hospital, Milan, Italy 3Effata Institute, Lucca, Italy

Article Info Abstract

Article Notes Today, it is known that all human biological functions are under two Received: April 1, 2020 fundamental regulatory systems, consisting of the endocrine system and the Accepted: April 21, 2020 cytokine network. Moreover, it has been shown that the released *Correspondence: from the activated immune cells do not influence only immune functions, but Paolo Lissoni, Institute of Biological Medicine, Milan, Italy; also the whole biological system, including the various metabolic activities, Email: [email protected]. the cardiovascular system, and the functionless of the neuroendocrine system itself. Unfortunately, despite the well-demonstrated importance of cytokines © 2020 Lissoni P. This article is distributed under the terms of in maintaining the status of health, from a clinical point of view the routine the Creative Commons Attribution 4.0 International License. evaluation of the cytokine system still remains unconsidered to establish Keywords the status of health, since it is investigated only in severe conditions, such as Cytokines septic shock, disseminated intravascular coagulation and respiratory distress, Cytokine network which have been demonstrated to be due to an abnormal endogenous Inflammation production of inflammatory cytokines, namely IL-6, TNF-alpha, and IL-1 beta. This clinical deficiency was depended on several factors, particularly on the complexity of cytokine interactions themselves, but also on the decision to use artificial molecules, such as monoclonal antibodies against the various cytokines, to counteract their eventual abnormally enhanced endogenous production, rather than to investigate the mechanisms responsible for their altered production and to correct eventual alterations. The main reason of the complexity of the cytokine network is related to the fact that the interactions occurring among the different cytokines are often founded on positive feedback mechanisms, then on reciprocal stimulatory actions, while the endocrine system is substantially based on negative feedback circuits. The aim of the present review is to propose a synthetic knowledge regarding the main effects and the source of origin of each single discovered up to now, to elaborate a first preliminary fundamental physiology of the cytokine network.

Introduction

As “cytokines”, we define a great group of small proteins produced by the immune cells after their activation, as well as by stromal cells, involved in the modulation of the biological immune-inflammatory response, and which are responsible for the pathogenesis of human systemic diseases. The cytokines more exactly produced by lymphocytes were defined with the term of . Finally, cytokines received their interleukin (IL) definition at the second International Lymphokinest Conference in Interlaken (Switzerland) in 1979. Since then, the number of interleukins has grown significantly. At the end of 21 century, the total group of interleukins has reached the number of 30, and kept on increasing more, and at present there are currently 40 interleukins named. Obviously, there are only few studies concerning the biological significance of the last discovered 10 interleukins. Moreover, since the last discovered interleukin in Page 18 of 28 Lissoni P, Messina G, Pelizzoni F, Rovelli F, Brivio F, Monzon A, Crivelli N, Lissoni A, Tassoni S, Sassola A, Pensato S, Di Fede G. The Fascination of Cytokine Journal of Infectiology Immunological Science. J Infectiology. 2020; 3(1): 14-28

1 its complete structure was IL-40 in 2017 , and no other a therapeutic point of view, in vivo it is more easy to act interleukin was discovered during the last two years, we by blocking an eventual exaggerated secretion of some may consider, at least from a synthetic and symbolic point cytokines by the administration of specific monoclonal of view, that the number of interleukins may be 40. Finally, antibodies against the single interleukin, whose endogenous some interleukins have received a great attention by the production has been proven to be abnormally increased in Immunologists, while others have been less investigated, each single pathology, such as the enhanced production3 of since they were considered to play a less importance role IL-6, TNF-alpha and TGF-beta in the 4metastatic and in the human biology. In any case, the classification of IL-17 in the autoimmune diseases . On the contrary, in interleukins themselves is still ambiguous, since there is the presence of an interleukin deficiency, such as that of no clear definition of interleukins with respect to that of IL-2 in the advanced neoplasms, the therapy will consist cytokines. Then, some cytokines provided by important of the administration of the interleukin, whose secretion biological effects are not defined as interleukins, for is abnormally low. Finally, the complexity of interleukin example -alpha (TNF-alpha) and system is furtherly amplified 7 by the fact that some8, transforming -beta (TGF-beta), which are interleukins 6may exist in different isoforms, such as IL-17 respectively characterized by proinflammatory, or by anti- in 6 isoforms , IL-36 in 4 isoforms , and IL-37 in 5 isoforms inflammatory effects. Today, it is known that the human as well as by the fact that some isoforms of interleukin may systemic diseases may be interpreted as the end-result display opposite effects. In vivo, there are two fundamental of an interaction between the action of some exogenous mechanisms to protect against an eventual excessive agents and host biological response. Therefore, the human secretion of some cytokines, consisting5 of the production9, which pathologies could be sub-divided into two major classes, of a soluble receptor, such as for IL-2 , or a binding protein consisting of diseases namely depending on the action (BP) for a specific interleukin, such as that for IL-18 of exogenous agents, including microbes and potential Classificationact by reducing itsof freeInterleukins amount in the blood. toxic or cancerogenic agents, and diseases mainly due to host immune-biological response. The human systemic diseases, including cancer, autoimmune pathologies and The interleukins may be classified according to three allergy, would depend at least in part on host biological main principles, consisting of their family of origin, which response, consisting of an exaggerated immune reaction, is related to their chemical structure, their effects on the such as autoimmune and allergic pathologies, or a deficient inflammatory response, by classifying interleukins in two , such as that occurring in the neoplastic main groups, respectively provided by proinflammatory or diseases. The endocrine mechanisms may explain some anti-inflammatory activities, and finally their activity on human pathologies, but the most severe human diseases tumor growth. As far as the origin family, it is possible to would be due to an altered immune function. Moreover, recognize 7 fundamental family of interleukins, as follows: while the endocrine system is regulated by negative 1) IL-1 family: this group includes IL-1 beta (since IL-1 feedback mechanisms, which may be understood on alpha is an intracellular protein), IL-18, IL-33, IL-36, IL-37, the basis of rationale considerations, the biology of the and IL-38; 2) IL-2 family: it contains IL-2 itself, IL-7, IL-15, cytokine network cannot be explained only in relation to and IL-21; 3) IL-4 family: it includes IL-4 and IL-13; 4) IL-6 a logical ratio, since most cytokine secretions are linked by family: it is consisting of IL-6 and IL-31; 5) IL-10 family: reciprocal stimulatory effects based on positive feedback it is constituted by several factors, as follows: IL-10, IL-19, mechanisms, as well as since some cytokines may induce IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29; 6) IL-12 family: the secretion of other cytokines provided by opposite it contains IL-12, IL-23, IL-27, IL-30, IL-35, and IL-39; 7) activities in an attempt to counteract their excessive effects IL-17 family: it is characterized by the 6 different isoforms on the biology of the living organisms. Therefore, we need of IL-17 itself. Finally, other interleukins are not classifiable a different mental approach with respect to the simple in a specific family of interleukins, including IL-3, IL-5, IL- mechanic logical ratio, also founded on symbolic archetypic cytokines9, IL-8, IL-11,1 IL-16, IL-25, IL-31, IL-32, IL-34, and IL-40. dynamics. In addition, because of the complex interactions The IL-12 family is unique in having the only heterodimeric among the different interleukins, as well as because the in, . On the other side, as far as the relation with vivo existence of a physiological neuroendocrine regulation2 their effect on the inflammatory status, interleukins may be of the cytokine network and interleukin secretion subdivided into proinflammatory and anti- effects observed in vitro may be different from interleukins. The group of the major proinflammatory those occurring in vivo, and this difference may represent interleukins includes IL-1 beta, IL-2, IL-6, IL-8, IL-12,IL-14, a limiting factor to understand the real impact of the single IL-15, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-26, interleukin in the pathogenesis of human diseases, as well IL-25, IL-28, IL-31, IL-32, IL-35, IL-36, and IL-38. On the as to establish the possible therapeutic use of each single other side, the group of the anti-inflammatory interleukins interleukin in the various pathologies, Therefore, from includes IL-7, IL-10, IL-19, and IL-38. In any case, it has

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appeared that some interleukins, such IL-2, and IL-12 within the group of the anti-inflammatory cytokines, such would be characterized by a dual proinflammatory and as IL-4 and IL-13, on the basis of their in vitro results, may anti-inflammatory actions. In more detail, particularly IL-2 in vivo present an inflammatory clinical behaviour because and IL-12, which are generally included within the group of of their stimulation of histamine secretion and the possible the inflammatory cytokines, may play both inflammatory induction of capillary leak syndrome. The classification of and anti-inflammatory effects. The inflammatory action cytokines in relation to their influence on the biological of IL-2 is mainly due to its stimulatory effect on the inflammatory response is reported in Table 1. Another system, whereas the anti-inflammatory one important question regards the actions of the different would depend on its inhibitory action on IL-17 secretion5 interleukins on T lymphocyte proliferation5 because its and on its promoting effect on TGF-beta secretion . On influence on the anticancer immunity . Most cytokines the other side, the inflammatory action of IL-12 would tend to exert both anti-tumor and pro-tumor activities, be linked to its ability to determine T naïve lymphocyte and at present, the only cytokine, which has been proven differentiation into TH1 cells, with a consequent enhanced to really enhance T lymphocyte count and to determine an IL-2 production, and to its inhibitory action on TGF-beta evident5 lymphocytosis14 in clinical15 studies16 still remains IL- secretion, while its anti-inflammatory effect10 is mainly 2 , even though IL-7 , IL-15 and IL-12 could also play depending on the inhibition of IL-17 secretion . IL-3 also a stimulatory action on T lymphocyte16 proliferation. IL-12 may play both inflammatory and anti-inflammatory11 effect, alone induces lymphocytopenia , while the association with the anti-inflammatory activity due to its ability to between IL-12 and IL-2 has17 been proven to paradoxically inhibit IL-2-induced macrophage activation . On the same determine the maximal lymphocytosis described up way, IL-11 may exert both pro- and anti-inflammatory to now in the literature . Finally, by considering their oneeffects, depending on the different experimental conditions, action on cancer development and proliferation in clinical but 12 the main activity would be the anti-inflammatory conditions, at present the only interleukin clearly provided17 . In any case, it is important to remark that there is by5 anticancer activity in humans still remains the only IL- no relation between interleukin family and its influence 2 , and also IL-12, but only in combination with IL-2 . In on the inflammatory response, since interleukins included addition, by simultaneously considering the effects on the within the same family may exert opposite effects on the inflammatory response and on tumor growth, it appears inflammatory status. Moreover, by considering that the that all clearly inflammatory cytokines tend to display a pro- inflammatory status is characterized by interactions tumoral action by suppressing the antitumor immunity. Not between pro- and anti-inflammatory cytokines, it has only,18 but it has appeared that the main cytokines provided to be taken into consideration that the classification of by anti-inflammatory action, namely TGF-beta and IL- interleukins in one or the other category would be too 10 , tends to play a pro-tumoral effect by suppressing simplistic, since a given cytokine may in some cases exert the antitumor immunity, with the only exception of IL-10, either pro- or anti-inflammatory13 effects, depending on the which, in addition to its immunosuppressive activity on different biological conditions . Inflammatory response both IL-2 and IL-12 secretion, as well as TGF-beta, could18 itself may determine the release of anti-inflammatory potentially stimulate the antigen-dependent cytotoxicity cytokines, such as IL-10, to control and counteract the mediated by the cytotoxic T lymphocytes (CD8+) . excessive inflammatory reaction. In addition, it has also Then, IL-10 would function as the point of equilibrium to be considered that the anti-inflammatory factors are between the immune surveillance due to the anticancer1 often represented by soluble cytokine receptors, such as immunity, and the suppression of the anticancer immunity. the soluble TNF-alpha receptors p55 and p75, as well as Unfortunately, as observed by Catalan-Dibene et al. , most IL-1 receptor antagonist IL-1ra 1 and 2, which reduce the interleukins were discovered by the older generation of free amount of a given cytokine, rather than by cytokines Immunologists, while in contrast the clinical investigation directly provided by anti-inflammatory effects. In any case, of the importance of cytokines in the pathogenesis of the main confusion is due to the discrepancy between in human systemic diseases and their treatment does not vitro and in vivo effects of each single cytokine, as shown seem to be considered as a fundamental topic by the by the fact that some interleukins generally included new generation of Immunologists. This evidence could Table1. Cytokine classification in relation to the effects on the biological inflammatory response. IL-1 beta, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, INFLAMMATORY CYTOKINES IL-25, IL-31, IL-32, IL-33, IL-36, IL-38, IL-39, IL-40, TNF-alpha IL-7, IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-27, IL-28, IL-29, IL-30, IL-34, IL-35, IL- ANTI-INFLAMMATORY CYTOKINES 37, TGF-beta INTERLEUKINS WITH DUAL INFLAMMATORY AND ANTI- IL-2, IL-3, IL-11, IL-12 INFLAMMATORY ACTION

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5 also be due at least in part to the fact that a complete cytotoxicity played by LAK cells is counteracted by IL-6 . comprehension of the single interleukin requires several Unfortunately, IL-2 may induce a concomitant5 stimulation knowledges, including its molecular chemical structure, of regulatory T lymphocytes (T reg) , with a consequent its receptors, and its biological activity in the different enhanced production of TGF-beta, which in contrast may experimental and clinical conditions. Then, from a clinical inhibit the antitumor immunity, by representing5 the most point of view, it would be enough to synthetically know the active endogenous immunosuppressive agent . In any main effects of each single interleukin and cytokine, at least case, before the recent development of artificial cancer on the inflammatory response, lymphocyte proliferation, immunotherapies with anti-PD1 or anti-PD-L1 and PD- and tumor cell proliferation, as well as its main source of L2, IL-2 therapy had represented the only real scientific production.Main Effects of the Single Interleukin immunotherapy of human neoplasms, which would have to be rediscovered and improved on the basis of the IL-1 beta successive knowledges concerning the physiopathology of the anticancer immunity.5 Not only, but IL-2, by stimulating TGF-beta secretion and by inhibiting that of IL-17 (21), IL-1 beta is produced by . It stimulates the could constitute an effective immunotherapy also for the production of IL-6 from macrophages themselves. Then, autoimmune diseases. Then, biological strategies, carried IL-6 is the main cytokine responsible for the induction of out to modulate TGF-beta response to IL-2, could make IL-2 the acute inflammatory response19 by stimulating the hepatic treatment an effective immunotherapy for both cancer and production of acute phase-related proteins, namely the IL-3autoimmune pathologies. C-reactive19 protein (CRP) . IL-1 beta would cooperate with IL-18 in inducing macrophage-mediated inflammatory response . IL-1 beta has been proven to induce19 high levels IL-3 is a multi-colony stimulating factor for granulocytes, of serum amyloid A, and to participate in association with macrophages, erythroid cells and mast cells, produced by IL-18 in the pathogenesis of atherosclerosis . IL-1 beta macrophages and stromal cells, and23 it could be involved would represent the main endogenous pyrogenic factor. in chronic inflammatory conditions . However, IL-311 has Moreover, it has been proven to stimulate cortisol secretion also been proven to counteract IL-2-induced macrophage during the inflammatory conditions to counteract a activation,IL-4 then to exert an anti-inflammatory activity . possible excessive reaction. Finally, it is also an19 anorectic cytokine more potent than leptin itself, then it would be IL-2responsible for cancer-related loss of body mass . IL-4 is a complex cytokine24 having opposite effects in both cancer and autoimmunity . It is mainly released by TH2 lymphocytes, and it represents on the same time the main IL-2 is produced by TH1 lymphocytes, whose differentiating factor of TH2 cell themselves. At present, it differentiation is stimulated20 by IL-12 released from the is known that the difference between TH1 and TH2 cells mature dendritic cells . IL-2 is perhaps the main cytokine is only functional, since no difference in the expression of of the whole immune system, by representing the main clusters of differentiation has been documented between cytokine responsible for T lymphocyte proliferation. In TH1 and TH2 cells. TH1 cells namely release IL-2 and fact, at the beginning of 13 its discovery, it was called as T gamma- (IFN), while TH2 cells may produce IL- cell growth factor (TCGF) . TH1 lymphocytes may secrete 4, IL-5, IL-6 and IL-10. As far as its possible influence on IL-2 and concomitantly express IL-2 receptor, with a cancer growth, despite the controversial results reported consequent proliferation of themselves. Then, TH1 would in the literature, at present IL-4 would seem to play a major constitute the only immune cells able to realize a self- pro-tumoral role, because of its stimulatory effect on TGF- cloning, by playing an essential13 role in the functionless beta secretion,25 and its amplification of IL-17-induced IL-6 of the whole immune system . The fundamental role of IL-5production . IL-2 in maintaining an effective immune functionless is demonstrated by the fact that AIDS-related IL-2 deficiency may allow a complete failure of the immune responses. IL-5 is the main growth factor for eosinophils. It is The antitumor immune action of IL-2 is substantially released5,23 from TH2 lymphocytes under stimulation by IL- due to its ability to induce the evolution of NK cells 2 . In fact, eosinophilia is one of the most typical biological5 into -activated killer (LAK) cells, which are responses to IL-2 administration in . capable of destroying fresh human 13 cancer cells through Eosinophils23 are mainly involved in the control of protozoan an antigen-independent cytotoxicity , while NK cells in parasites , but it could display a low anticancer5 activity, their basal status are unable to kill fresh human cancer at least under IL-2 cancer immunotherapy . IL-3, IL-5, and cells, but only laboratory cancer cell lines. The antitumor GM-CSF have been shown to present a common beta chain. Page 21 of 28 Lissoni P, Messina G, Pelizzoni F, Rovelli F, Brivio F, Monzon A, Crivelli N, Lissoni A, Tassoni S, Sassola A, Pensato S, Di Fede G. The Fascination of Cytokine Journal of Infectiology Immunological Science. J Infectiology. 2020; 3(1): 14-28

IL-6 cytokines and it acts32 in synergistic combination with other IL-6 is one of the main inflammatory cytokines, produced . In addition, it has also appeared to stimulate by macrophages stimulated by IL-1 beta, as well by TH2 megakaryocyte proliferation, by acting as a potential32 growth cells. Released under IL-1 beta stimulation, IL-6 induces the factor for platelets to increase platelet count . Finally, IL- hepatic production of acute phase proteins. IL-6 exerts a 11 has been proven to stimulate the osteoclastogenesis,12 with a consequent bone loss and osteoporosis . pro-tumoral immunosuppressive activity5 by counteracting IL-12 IL-2-induced activation of LAK cells . Moreover, IL-6 would be the main cytokine responsible26 for septic shock-related untreatable hypotension . IL6 would also exert a partial IL-12, which is produced by dendritic cells and by IL-7stimulatory action on platelet generation. macrophages when they assume the function of antigen presenting cells, may be considered as the link between innate and acquired immunity. Its main function is the IL-7 is mainly produced by stromal cells, and it has stimulation of the differentiation into TH1 lymphocytes,20 appeared14 to be required for development and with the consequent production of IL-2 and gamma-IFN ,. survival . In more detail, IL-7 would be essential for In association with IL-2, it represents the main anticancer5,20 the transformation of the double negative CD4 and CD8 cytokine in humans through several mechanisms thymocyte progenitors into double positive CD4 and including direct activation of antigen-dependent CD8 thymocytes in the thymus (27). Finally, despite the anticancer immunity mediated by cytotoxic T lymphocytes, controversial results, IL-7 would exert27 a major anti- stimulation of IL-2 secretion from TH1 cells and inhibition IL-8inflammatory and anti-tumor activities . of the immunosuppressive activity of TGF-beta, the main endogenous immunosuppression factor on the anticancer immunity.IL-13 IL-8 is a like-IFN cytokine, produced by macrophages and stromal cells, with antiviral, inflammatory, angiogenic and pro-tumoral activities. The main target for IL-8 action IL-13 is mainly produced by TH2 lymphocytes and would be the neutrophils,28 by stimulating their proliferation basophils,24,25 and its effects are like to those exerted by IL- and chemotactic activity . High levels of IL-8 have been IL-144 . proven to predict a negative prognosis either in cancer, or inIL-9 infectious diseases. IL-14 is one of the main growth factors (BCGF). Then, it could play a stimulatory33 effect on the proliferation IL-9 is a still less investigated cytokine. It is mainly IL-15of hematologic malignancies . released from TH2 cells, and it would be involved in the pathogenesis of cancer, namely in malignant lymphoma, since high serum levels29 of IL-9 have appeared to predict IL-15 exerts immunological effects comparable to those15 a negative prognosis . IL-9 would inhibit the anticancer of IL-2, with stimulation of T, B and NK cell proliferation . immunity by stimulating TGF-beta production from T30 reg In addition, IL-1515 may induce effector memory cytotoxic cells, as well as that of IL-17 from TH17 lymphocytes . As T lymphocytes . Preliminary clinical results would far as its effects on the inflammatory 31 status, IL-9 would suggest that IL-15 may represent a new strategy in the exertIL-10 a predominant inflammatory role . immunotherapy of cancer, with results comparable15 to those exerted by IL-2, but with less side-effects . There are two isoforms of IL-15, which is mainly produced by , IL-10, namely produced by TH2 lymphocytes,18 T reg cells, macrophages and dendritic cells, even though it may be and macrophages, is one of the main anti- inflammatory producedIL-16 by several tissues. and immunosuppressive endogenous factors . It would be also involved in B cell maturation. Despite its inhibitory effect of the anticancer immunity, it could be able18 of some IL-16 is a still less investigated cytokine, characterized anti-tumoral activity by stimulating the action of cytotoxic by several and often opposite functions, and provided by IL-11T lymphocytes and their immunological memory . inflammatory effects34 due to a stimulation of the - macrophage system . Then, it is involved as a mediator of the inflammatory processes. In 34addition, it has also been IL-11 is a multifunctional hematopoietic cytokine, proven to inhibit HIV replication . produced mainly by stromal cells under IL-1 stimulation,

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IL-17

Despite the controversial data of the literature, IL-19 would play a prevalent anti-inflammatory action. Moreover, IL-43 IL-17 is produced by TH17 lymphocytes, and it seems 19 has been shown to play an atheroprotective role . to constitute the main cytokine responsible for the onset In contrast, IL-20 and IL-24 would exert a predominant of autoimmune mechanisms, since its main activity would inflammatoryIL-20 activity. consist of the inhibition of T17 reg cells, with a consequent diminished inhibitory control on the activation of autoreactive T lymphocytes . The control of IL-17 is very complex, since it has appeared to be under a stimulatory IL-20, as well as IL-19 and IL-24, would be involved in the mechanism played10 by IL-1 beta and IL-6,5,35 while it is namely pathogenesis of the autoimmune diseases by stimulating inhibited by IL-12 , as well as by IL-2 . More17 complex is the release of inflammatory cytokines, even though it is still the interaction between IL-17 and TGF-beta, since IL-17 unclear whether it may be one of their causes,42.44 or the simple inhibits TGF-beta secretion from T reg cells , while the IL-21reaction to counteract their progression . effects of TGF-beta on IL-17 secretion are depending on the concentrations of some other cytokines. In more detail, TGF-beta alone would play a major inhibitory action on IL-21, which is produced by TH1 lymphocytes, has IL-17 secretion, 36 while it exerts37 a stimulatory role in the appeared to be a potentially effective antitumor cytokine, presence of IL-6 or IL-23 . Therefore, IL-23 would also as well as IL-12. The antitumor activity of IL-21 seems to be involved as well as38 IL-17 in the pathogenesis of the be due to its inhibitory action on T reg cells and on TH17 autoimmune diseases . As far as the possible influence of cell differentiation. Moreover, IL-21 in association with IL-17 on cancer is concerned, the inhibitory effect of IL-17 IL-12 may display an inhibitory effect on T reg and TH17 on the immunosuppressive action of T reg cells could be lymphocytes superior45,46 to that observed with IL-21 alone a potentially therapeutic effect in cancer, but it would be and IL-12 alone . IL-21 has also appeared to abrogate47 abrogated by the concomitant direct39 stimulatory action of the increase in T reg cells induced by IL-2 plus TGF-beta . IL-17 on cancer cell proliferation . There are six isoforms Then, IL-21 could be effective in the immunotherapy of of IL-17 (A, B, C, D, E, F), and the most biologically active cancerIL-22 in association either with IL-12, or IL-2. IL-18form is IL-17A.

IL-22 is produced by several types48 of lymphocytes, IL-18 is a pleiotropic cytokine produced by several including TH1, TH17 and NK cells . IL-22 plays a pro- cells, including macrophages, hematopoietic cells and inflammatory effect by acting synergistically with TNF- endothelial cells. It is involved in the regulation of innate alpha, IL-1 beta and IL-17, but also by determining some and acquired immunity,9,40 as well as IL-12, even though with unique effects through a direct stimulation of acute phase different effects . In more detail, as well as IL-12 and IL- reactantIL-23 production. 15, IL-18 is a potent inducer of gamma-IFN production by both NK cells and TH1 lymphocytes, and it would be41 involved in the pathogenesis of the autoimmune diseases . IL-23 may be secreted by several lymphocytes, and In fact, the autoimmune diseases are often characterized its main effect is constituted by the stimulation of TH17 by a concomitant increase in both IL-18 and gamma- activation and IL-1749 production, either alone, or in IFN blood concentrations. On the contrary, the allergic association with IL-6 . Then, IL-23 would play an important50 pathologies tend to be characterized by high levels9,41 of IL- roleIL-24 in the development of the autoimmune diseases . 18 in association with low levels of gamma-IFN . IL-18 would also exert an angiogenic action. Finally, IL-18 has also been proven to stimulate the41 appetite and to play a IL-24 is produced by TH2 cells and monocytes. It has roleIL-19 in the development of obesity . appeared to stimulate TNF-alpha and IL-6 secretion. Then,51 IL-25it is involved in determining the inflammatory processes .

IL-19 is42 a member of IL-10 family, as well as IL-20, IL-22, 52 and IL-24 . IL-19 is namely produced by TH2 lymphocytes, The past definition of IL-25 was IL-17E . It plays a as well as IL-20, IL-22, IL-24. High levels of IL-19, IL-20, unique function within IL-17 cytokine family. IL-25 is and IL-24 have been described in autoimmunity, but produced by several cells, including TH2 lymphocytes, it remains to be established whether they may play an mast cells, and macrophages. It stimulates TH2 functions, inflammatory, or an anti-inflammatory action to counteract with a consequent enhanced production of IL-4, IL-5, and autoimmunity-related inflammatory status, such as IL-10. IL-13. Moreover, IL-25 has also been proven to stimulate IL-

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IL-32

17 secretion from TH17 lymphocytes. Then, it would play a role in the onset of allergic inflammation, including asthma. IL-32 may be produced by several peripheral blood1 In addition, it would be also52 involved in the pathogenesis of mononuclear cells (PBMCs), and there are various isoforms IL-26the autoimmune diseases . of IL-32, probably provided by different functions . IL- 32 stimulates the production of several inflammatory cytokines, including TNF-alpha, IL-1 beta, IL-6, and several IL-26 is produced by both epithelial and immune cells, , then it plays an important1 inflammatory including TH1, TH17, and NK cells. It may exert antiviral activity 60 and would be involved in several autoimmune and antimicrobial effects. It53 would be also involved in the diseases , as well as in cancer progression . Therefore, IL- IL-27chronicity of inflammation . 32 may be considered as an inflammatory and pro-tumoral cytokine.IL-33

The main source of IL-27 is represented by the antigen presenting cells,54 then the dendritic cells and the activated IL-33 is a member of IL-1 family, and it is produced macrophages . IL-27 was initially thought to be a pro- by TH2 cells, eosinophils, basophils and mast cells. IL-35 inflammatory cytokine, but now it is known that it may play stimulates IL-4, IL-5 and IL-13 secretion, and it may allow an immunoregulatory role with anti-inflammatory54 activity eosinophilia and an increased secretion61 of IgE, then it is by inhibiting IL-17A and IL-2 secretion . Moreover, it has IL-34involved in the allergic disorders . also been proven to stimulate IL-10 secretion. Then, it could display a therapeutic role in the autoimmunity, but not of IL-28cancer, because of IL-10-induced immunosuppression. IL-34 is produced by macrophage and T reg cell systems, by keratinocytes in the skin and neurons in the IL-28, produced by dendritic cells, TH17, and - brain. IL-34 has been proven to interact with monocyte-, colony stimulation factor (M-CSF) in several functions,62 infected cells, has been proven55 to play an anti-viral activity IL-29by inhibiting viral replication . including the promotion of the osteoblastogenesis then it could deserve a potential therapeutic activity in osteoporosis. On the contrary,63 IL-11 has appeared to stimulate the osteoclastogenesis . Then, bone metabolism IL-29 is mainly produced by dendritic cells, monocytes would also be under a cytokine regulation, and IL-34 and and TH17 lymphocytes.56 As well as IL-28, it is a cytokine with , IFN-like activity . Then, it may inhibit viral replication with IL-11 would constitute a functional axis with opposite64 mechanisms like to those of IFN-alpha. It exerts an anti- effects. In addition, IL-34 may stimulate T reg cell system inflammatory effect in the allergic diseases. In contrast, its with a consequent induction of an immunosuppressive role in cancer is still controversial, since either pro-tumoral status, which may prevent acute rejection after organ andIL-30 anti-tumoral activities have been described. transplantation, but on the same time it may promote tumor progression. Finally, IL-34 may act as a profibrotic factor, then it would be involved65 in age-related organ IL-30, represented by the p28 subunit of IL-27, displays fibrosis,IL-35 as well as TGF-beta . an anti-inflammatory57 activity like to IL-27 by inhibiting IL-17A production . Moreover, it has been recently 1 demonstrated that IL-30 expression may predict a negative IL-35 was discovered since 1997, but not named IL-35 prognosis at least in some1,58 tumor histotypes, including until 2017 . IL-35 is mainly produced by T reg cells and IL-31breast and prostate tumors . regulatory B lymphocytes, and it exerts a strong suppressive activity on several immune functions by activating T reg

1 cell system, with a consequent enhanced production of IL-31 is a member of IL-6 family, then it is a 4-helix both TGF-beta and IL-10, 66and a following inhibition of TH1 bundle cytokine . IL-31 is produced by TH2 lymphocytes, cells and IL-2 production . Moreover, IL-3566 has also been as well as by eosinophils, and it acts on IL-31 receptor A shown to inhibit TH17 cell proliferation , then it could be and receptor. IL-31 secretion correlates with more effective than TGF-beta and IL-10 themselves in the that of two other TH2 cytokines, IL-4 and IL-13, and it treatment of . On the contrary, IL-35- seems to be involved in .59 Moreover, IL-31 induced stimulation of T reg cell system might negatively correlates with the intensity of pruritus . influence the prognosis of human tumors.

Page 24 of 28 Lissoni P, Messina G, Pelizzoni F, Rovelli F, Brivio F, Monzon A, Crivelli N, Lissoni A, Tassoni S, Sassola A, Pensato S, Di Fede G. The Fascination of Cytokine Journal of Infectiology Immunological Science. J Infectiology. 2020; 3(1): 14-28

IL-36

several cell types, including skin, thymus, tonsil, liver and brain, and provided by a typical pro-inflammatory IL-36 is a proinflammatory cytokine produced by naïve activity by stimulating the secretion of several other pro- CD4 T cells, skin keratinocytes, myeloid cells, Langerhans inflammatory cytokines,69 including IL-1 beta, IL-22, IL-17A cells, and mucosal . Its proinflammatory role and IL-36 itself . is due to a stimulating effect on the secretion of most IL-39 inflammatory67 cytokines, including TNF-alpha, IL-6, IL-17A and IL-23 . However, in contrast to the behaviour of other inflammatory cytokines, which play a major inhibitory role IL-3970 is the most recently discovered member of IL-12 on IL-2 secretion, IL-36,67 as well as IL-12, may also promote1 family . It is mainly secreted by activated B lymphocytes, TH1 differentiation . In any case, IL-36 would play an dendritic cells and macrophages. It mediates the essentialIL-37 role in skin defense and tissue repair in the gut . inflammatory response by promoting the differentiation of IL-40neutrophils and their chemotactic ability.

1 IL-37 is also an important cytokine of the IL-1 family, with structural similarities with IL-18 (1), which is present IL-40 is the last discovered interleukin , produced by in five isoforms (IL-37 a, b, c, d, e), but it is paradoxically activated B cells, bone marrow cells and some tissues, such provided by an anti-inflammatory activity, since it has as the mammary gland. It plays an important role1 in normal been proven to completely suppress the secretion of the B cell function, particularly for IgA secretion at local level. The action of71 IL-40 is enhanced by TGF-beta , as well as main pro-inflammatory cytokines, including TNF-alpha,68 previously observed for other stimulating factors for B IL-6, and IL-1 beta, as well as several chemokines . Then, lymphocytesThe similarity. among the Different Interleukins IL-37 is an important cytokine in immune homeostasis as an immune regulator, with potential therapeutic benefits in the immunotherapy of cancer by blocking the According to the biological behaviour existing in immunosuppressive action of IL-6, IL-1beta and TNF-alpha, Nature, several biological functions are present in a double as well as of autoimmune pathologies by counteracting similar forms to compensate possible deficiencies of the inflammatoryIL-38 cytokine-induced tissue damage. single form. Then, even though in a schematic way, the 40 interleukins could be subdivided in 20 fundamental couples characterized by similar and complementary IL-38 is another member of IL-1 family, produced by functions. As shown in Table 2, it is possible to identify Table 2. The 20 functional couples of the 40 human interleukins. COUPLE OF INTERLEUKINS SIMILAR FUNCTIONS IL-1 – IL-18 * Induction of the acute inflammatory response IL-2 – IL-12*+ Stimulation of antigen-independent and- dependent anticancer immunity IL-3 - IL-7 + Development of hematopoietic lymphocyte and monocyte systems IL-4 – IL-13 * Stimulation of histamine release IL-5 – IL-25* Stimulation of eosinophil generation and functions IL-6 – IL-22* Activation of the acute inflammatory reaction IL-8 – IL-39 * Activation of neutrophil functions IL-9 – IL-32 *+ Predisposition to autoimmunity and cancer IL-10 – IL-35 + Immunosuppression of IL-2-dependent immunity, including the anticancer one IL-11 – IL-34 + Regulation of bone metabolism IL-15 – IL-21 * + Cooperation with IL-2 and IL-12 in the induction of anticancer immunity IL-14 – IL-40 * Stimulation of B lymphocytes IL-16 –IL-26 * Anti-viral activity IL-17 – IL-23 * Activation of TH17 system in inhibiting T reg cell functions IL-19 – IL-37 + Stimulation of TGF-beta production and inhibition of inflammatory cytokines IL-20 – IL-24* Stimulation of inflammatory cytokine TNF-alpha and IL-6 production IL-27 – IL-30 + Anti-inflammatory action by inhibiting IL-17 cell system IL-28 – IL-29 * Anti-viral activity IL-31 – IL-33 * Stimulation of IL-4 and IL-13 secretion IL-36 – IL-38 * Stimulation of IL-6 and TNF-alpha secretion * Inflammatory cytokines; + Anti-inflammatory cytokines; *+ Dual effects on the inflammatory response

Page 25 of 28 Lissoni P, Messina G, Pelizzoni F, Rovelli F, Brivio F, Monzon A, Crivelli N, Lissoni A, Tassoni S, Sassola A, Pensato S, Di Fede G. The Fascination of Cytokine Journal of Infectiology Immunological Science. J Infectiology. 2020; 3(1): 14-28

Table 3. Main biological effects of the 40 interleukins and their site of production. INTERLEUKIN SITES OF ORIGIN MAIN IMMUNOBIOLOGICAL FUNCTIONS IL-1 beta Macrophages Stimulation of IL-6 secretion, fever and interactions with IL-18 IL-2 TH1 cells Lymphocytosis, eosinophilia, LAK cell generation IL-3 Macrophages, stromal cells Multi-colony stimulating factor IL-4 TH2 cells, basophils Stimulation of histamine release IL-5 TH2 Stimulation of eosinophil generation IL-6 Macrophages Stimulation of CRP hepatic production, severe hypotension IL-7 Thymic stromal cells T lymphocyte development and differentiation IL-8 Macrophages, stromal cells Like IFN-activity IL-9 TH2 cells Stimulation of T reg and TH17 lymphocytes IL-10 TH2 cells, macrophages Inhibition of IL-2 and IL-12 secretions IL-11 Stromal cells Stimulation of platelet generation, osteoclast cell stimulation IL-12 Dendritic cells Stimulation of TH1 cells, inhibition of T reg and TH17 cells IL-13 TH2 cells IL-4-like activity IL-14 Macrophages Growth factor for B lymphocytes IL-15 Dendritic cells, macrophages Stimulation of T cells, B cells and NK cells IL-16 Macrophages, stromal cells Macrophage stimulation, inhibition of HIV replication IL-17 TH17 lymphocytes Inhibition of T reg cells, reciprocal stimulation with IL-6 IL-18 Macrophages, endothelium Inflammatory activity in cooperation with IL-1 beta IL-19 TH2 lymphocytes Anti-atherosclerotic activity IL-20 Activated T lymphocytes Stimulation of inflammatory cytokine secretion IL-21 TH1 lymphocytes Inhibition of T reg cells secretion and TH17 differentiation IL-22 TH1, TH17, NK lymphocytes Stimulation of TNF, IL-1, IL-17 and CRP production IL-23 Several lymphocyte types Stimulation of IL-17 secretion IL-24 TH2 cells, monocytes Stimulation of TNF-alpha and IL-6 secretion IL-25 TH2, monocytes, mast cells Stimulation of IL-4, IL-5, IL-13 and IL-17 secretion IL-26 TH1, TH17, NK, epithelial cells Antiviral and antimicrobial activity IL-27 Dendritic cells, macrophages Inhibition of IL-17 and IL-2 secretion IL-28 Dendritic cells, TH17 cells Antiviral activity IL-29 Dendritic cells, TH17 cells IFN-like activity IL-30 Several immune cells Inhibition of IL-17 secretion IL-31 TH2 cells, eosinophils Stimulation of IL-4 and IL-13 secretion IL-32 PBMC Stimulation of TNF-alpha, IL-6 and IL-1 beta secretion IL-33 TH2, basophils, eosinophils Eosinophilia and increased IgE production IL-34 T reg cells, macrophages Interaction with M-CSF, osteoblastogenetic activity IL-35 T reg cells, B reg lymphocytes Stimulation of T reg cell activity, inhibition of IL-2 secretion IL-36 Naïve CD4+cells, keratinocytes Stimulation of TNF-alpha, IL-6, IL-17 and IL-23 secretion IL-37 Several immune cells Anti-inflammatory role inhibiting TNF, IL-6 and IL-1 release IL-38 Several cell types Stimulation of IL-1 beta, IL-22 and IL-17 secretion IL-39 B lymphocytes, dendritic cells Stimulation of neutrophil differentiation and chemotaxis IL-40 B cells and several tissue cells Stimulation of B lymphocytes * Inflammatory ILs: IL-1, IL-6,IL-8, IL-17, IL-18, IL-20, IL-22, IL-23, IL-24, IL-25, IL-32, IL-36, IL-38 * Anti-inflammatory ILs: IL-10, IL-35, IL-37 (plus TGF-beta) Dual effects: IL-2, IL-12, IL-3, IL-4, IL-5, IL-11, IL-13 + Antitumor ILs: clinically proven antitumor ILs: IL-2, IL-12, IL-15; possible antitumor ILs: IL-7, IL-21,IL-37 + ProtumorILs: IL-1, IL-6,IL-8,IL-10,IL-14,IL-17, IL-18,IL-20,IL-22,IL-23,IL-24,IL-25, IL-32, IL-35, IL-36, IL-38 (plus TGF-beta)

12 pro-inflammatory couples of interleukins (n=24), dynamics involved in regulating cytokine secretions is 5 anti-inflammatory couples of interleukins (n=10), beyond the simple mechanic logical reasoning, and, which it is and 3 couples of interleukins with dual pro- and anti- more similar to the symbolic imagination of the Tree72 of Life, inflammatory activities (n=6). Table 3 shows the principal the so-called Sephirot of the cabalistic tradition immunobiological effects of each single interleukin, its could be useful for meditation and for the identification of major source of origin, and its main proven interactions possible analogic connection occurring among the different with the other cytokines. It clearly appears that the cytokine secretion. Page 26 of 28 Lissoni P, Messina G, Pelizzoni F, Rovelli F, Brivio F, Monzon A, Crivelli N, Lissoni A, Tassoni S, Sassola A, Pensato S, Di Fede G. The Fascination of Cytokine Journal of Infectiology Immunological Science. J Infectiology. 2020; 3(1): 14-28

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